Establish a fast method to identify spermine and spermidine in biological samples and foods by matrix- assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS)

• Main Authors: Huai-Hsin Su, 蘇慧欣
• Other Authors: Chi-Yu Lu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/93814372089081822686

碩士 === 高雄醫學大學 === 生物化學研究所 === 99 === Polyamines consist of two or more primary amino structures, and are organic compounds containing aliphatic nitrogens. These organic compounds widely spread in animals and plants, and play an important role in the biological organisms. Spermidine and spermine are members of polyamine, their precursor is ornithine, derived from foods, intestinal normal flora, and de novo biosynthesis. Spermidine and spermine participate in the intestinal health and organ development. Currently, overexpression of spermidine and spermine had been reported associating to cell proliferation and tumor growth. There are several methods to analyze polyamines such as chromatography, radioimmunoassay, electrophoresis and enzymatic assay. As we known, this study is the first to introduce MALDI-TOF MS for monitoring spermidine and spermine in biological samples and foods without derivatization reaction. For sample preparation, saturated sodium hydroxide (NaOH) was used to alkalize the sample solution then ethyl acetate was utilized for spermine and spermidine extraction. Then the supernatants were spotted on the target plate and mixed well with the matrix, 2-mercaptobenzothiazole (2-MBT). The limit of quantitation (LOQ) and limit of detection (LOD) for spermidine and spermine analyses were 0.1μg/mL and 0.02μg/mL, the relative standard deviation (RSD) and relative error (RE) for intra- and inter-day analyses were below 20％. This proposed method was further applied to quantify spermidine and spermine in foods, human urine and blood samples. Documented sdudies showed that excess spermidine and spermine in the embryonic trophoblast cells (SGHPL-4 cells) could lead to apoptosis. Hence, spermidine and spermine were used to treat NRK-52E cells (from rat, kidney) and difluoromethylornithine (DFMO) was used as the inhibitor. The differences of apoptosis-related proteins were detected and compared by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).